As indicated above, the MN gene and protein are known by a number of alternative names, which names are used herein interchangeably. The MN protein was found to bind zinc and have carbonic anhydrase (CA) activity and is now considered to be the ninth carbonic anhydrase isoenzyme—MN/CA IX or CA IX [Opavsky et al., Genomics, 33: 480-487 (1996)]. According to the carbonic anhydrase nomenclature, human CA isoenzymes are written in capital roman letters and numbers, whereas their genes are written in italic letters and arabic numbers. Alternatively, “MN” is used herein to refer either to carbonic anhydrase isoenzyme IX (CA IX) proteins/polypeptides, or carbonic anhydrase isoenzyme 9 (CA9) gene, nucleic acids, cDNA, mRNA etc. as indicated by the context.
The MN protein has also been identified with the G250 antigen. Uemura et al. [J. Urol. 157 (4 Suppl.): 377 (Abstract 1475; 1997)] states: “Sequence analysis and database searching revealed that G250 antigen is identical to MN, a human tumor-associated antigen identified in cervical carcinoma (Pastorek et al., 1994).”
Zavada et al., International Publication No. WO 93/18152 (published Sep. 16, 1993) and U.S. Pat. No. 5,387,676 (issued Feb. 7, 1995) describe the discovery of the MN gene and protein. The MN gene was found to be present in the chromosomal DNA of all vertebrates tested, and its expression to be strongly correlated with tumorigenicity. In general, oncogenesis may be signified by the abnormal expression of MN/CA IX protein. For example, oncogenesis may be signified: (1) when MN/CA IX protein is present in a tissue which normally does not express MN/CA IX protein to any significant degree; (2) when MN/CA IX protein is absent from a tissue that normally expresses it; (3) when CA9 gene expression is at a significantly increased level, or at a significantly reduced level from that normally expressed in a tissue; or (4) when MN/CA IX protein is expressed in an abnormal location within a cell. WO 93/18152 further discloses, among other MN-related inventions, MN/CA IX-specific monoclonal antibodies (MAbs), including the M75 MAb and the VU-M75 hybridoma that secretes the M75 MAb. The M75 MAb specifically binds to immunodominant epitopes on the proteoglycan (PG) domain of the MN/CA IX proteins.
Zavada et al., International Publication No. WO 95/34650 (published Dec. 21, 1995) provides in FIG. 1 the nucleotide sequences for a full-length MN cDNA [also provided herein in FIG. 6 (SEQ ID NO: 1)] clone isolated as described therein, and the amino acid sequence [also provided herein in FIG. 6 (SEQ ID NO: 2)] encoded by that MN cDNA. WO 95/34650 also provides in FIG. 6 the nucleotide sequence for the MN promoter [SEQ ID NO: 24]. Those MN cDNA, promoter and amino acid sequences are incorporated by reference herein.
Zavada et al., International Publication No. WO 03/100029 (published Dec. 4, 2003) discloses among other MN-related inventions, MN/CA IX-specific MAbs that are directed to non-immunodominant epitopes, including those on the carbonic anhydrase (CA) domain of the MN/CA IX protein. An example of such a MN/CA IX-specific MAb is the V/10 MAb, secreted from the V/10-VU hybridoma.
The MN protein is now considered to be the first tumor-associated carbonic anhydrase isoenzyme that has been described. The carbonic anhydrase family (CA) includes eleven catalytically active zinc metalloenzymes involved in the reversible hydration-dehydration of carbon dioxide: CO2+H2OHCO3−+H+. CAs are widely distributed in different living organisms. The CAs participate in a variety of physiological and biological processes and show remarkable diversity in tissue distribution, subcellular localization, and biological functions [Parkkila and Parkkila, Scand J Gastroenterol., 31: 305-317 (1996); Potter and Harris, Br J Cancer, 89: 2-7 (2003); Wingo et al., Biochem Biophys Res Commun. 288: 666-669 (2001)]. Carbonic anhydrase IX, CA IX, is one of the most recently identified isoenzymes [Opavsky et al., Genomics, 33: 480-487 (1996); Pastorek et al., Oncogene, 9: 2877-2888 (1994)]. Because of the CA IX overexpression in transformed cell lines and in several human malignancies, it has been recognized as a tumor-associated antigen and linked to the development of human cancers [Zavada et al., Int. J. Cancer, 54: 268-274 (1993); Liao et al., Am. J. Pathol., 145: 598-609 (1994); Saarnio et al., Am Pathol, 153: 279-285 (1998)].
MN/CA IX is a glycosylated transmembrane CA isoform with a unique N-terminal proteoglycan-like extension. Through transfection studies it has been demonstrated that MN/CA IX can induce the transformation of 3T3 cells [Opavsky et al., Genomics, 33: 480-487 (1996); Pastorek et al., Oncogene, 9: 2877-2888 (1994)].
The MN protein was first identified in HeLa cells, derived from a human carcinoma of cervix uteri. Many studies, using the MN-specific monoclonal antibody (MAb) M75, have confirmed the diagnostic/prognostic utility of MN in diagnosing/prognosing precancerous and cancerous cervical lesions [Liao et al., Am. J. Pathol., 145: 598-609 (1994); Liao and Stanbridge, Cancer Epidemiology Biomarkers & Prevention, 5: 549-557 (1996); Brewer et al., Gynecologic Oncology 63: 337-344 (1996)]. Immunohistochemical studies with the M75 MAb of cervical carcinomas and a PCR-based (RT-PCR) survey of renal cell carcinomas have identified MN expression as closely associated with those cancers and confirm MN's utility as a tumor biomarker [Liao et al., Am. J. Pathol., 145: 598-609 (1994); Liao and Stanbridge, Cancer Epidemiology, Biomarkers & Prevention, 5: 549-557 (1996); McKiernan et al., Cancer Res. 57: 2362-2365 (1997)]. In various cancers (notably uterine cervical, ovarian, endometrial, renal, bladder, breast, colorectal, lung, esophageal, head and neck and prostate cancers, among others), MN/CA IX expression is increased and has been correlated with microvessel density and the levels of hypoxia in some tumors [Koukourakis et al., Clin Cancer Res, 7: 3399-3403 (2001); Giatromanolaki et al., Cancer Res, 61: 7992-7998 (2001)].
In tissues that normally do not express MN protein, MN/CA IX positivity is considered to be diagnostic for preneoplastic/neoplastic diseases, such as, lung, breast and cervical precancers/cancers [Swinson et al., J Clin Oncol, 21: 473-482 (2003); Chia et al., J Clin Oncol, 19: 3660-3668 (2001); Loncaster et al., Cancer Res, 61: 6394-6399 (2001)], among other precancers/cancers. Very few normal tissues have been found to express MN protein to any significant degree. Those MN-expressing normal tissues include the human gastric mucosa and gallbladder epithelium, and some other normal tissues of the alimentary tract. Paradoxically, MN gene expression has been found to be lost or reduced in carcinomas and other preneoplastic/neoplastic diseases in some tissues that normally express MN, e.g., gastric mucosa.
MN Regulation Under Hypoxia and Normoxia
Strong association of MN/CA IX with a broad range of tumors is principally related to its transcriptional regulation by hypoxia and high cell density, which appear to activate the MN/CA9 promoter through two different, but interconnected pathways [Wykoff et al., Cancer Res., 60: 7075-7083 (2000); Lieskovska, et al., Neoplasma, 46: 17-24 (1999); Kaluz et al., Cancer Res., 62: 4469-4477 (2002)]. Those two pathways are activated via stabilization of HIF-1α by hypoxia, and direct stimulation of MN/CA IX protein expression by the phosphotidylinositol-3-kinase (PI3K) pathway, respectively.
Hypoxia is a reduction in the normal level of tissue oxygen tension. It occurs during acute and chronic vascular disease, pulmonary disease and cancer, and produces cell death if prolonged. Pathways that are regulated by hypoxia include angiogenesis, glycolysis, growth-factor signaling, immortalization, genetic instability, tissue invasion and metastasis, apoptosis and pH regulation [Harris, A. L., Nature Reviews, 2: 38-47 (January 2002)].
The central mediator of transcriptional up-regulation of a number of genes during hypoxia is the transcription factor. HIF-1 is composed of two subunits: a constitutively expressed HIF-1β and a rate-limiting HIF-1α, which is regulated by the availability of oxygen. Under hypoxia, HIF-1α skips modification of its conserved proline and asparagine residues by oxygen-sensitive hydroxylases, thus avoiding degradation mediated by pVHL and inactivation mediated by FIH-1 (factor inhibiting HIF-1) [Maxwell et al., Nature, 399: 271-275 (1999); Jaakkola et al., Science, 292: 468-472 (2001); Ivan et al., Science, 292: 464-468, 2001; Jaakkola, et al., Science, 292: 468-472 (2001); Mahon, et al., Genes Dev., 15: 2675-2686 (2001)]. This leads to HIF-1α accumulation, dimerization with HIF-1β, binding to hypoxia response element (HRE) sites in the target genes, interaction with the cofactors and stimulation of the HIF-1 trans-activation capacity.
In the absence of oxygen, HIF-1 binds to HIF-binding sites within HREs of oxygen-regulated genes, thereby activating the expression of numerous hypoxia-response genes, such as erythropoietin (EPO), and the proangiogenic growth factor vascular endothelial growth factor (VEGF). In addition, HIF-1α can be up-regulated under normoxic conditions by different extracellular signals and oncogenic changes transmitted via the PI3K and MAPK pathways [Semenza, Biochem. Pharmacol., 64: 993-998 (2002); Bardos and Ashcroft, BioEssays, 26: 262-269 (2004)]. Whereas PI3K activation results in an increased level of HIF-1α protein, MAPK activation improves its trans-activation properties [Laughner, et al., Mol. Cell. Biol. 21: 3995-4004 (2001); Richard et al., J. Biol. Chem., 274: 32631-32637 (1999)].
MN/CA IX was shown to be one of the most strongly hypoxia-inducible proteins, via the HIF-1 protein binding to the hypoxia-responsive element of the MN promoter [Wykoff et al., Cancer Res, 60: 7075-7083 (2000); Svastova et al., Exp Cell Res, 290: 332-345 (2003)]. Like other HIF-1-regulated genes, the transcription of the MN gene is negatively regulated by wild-type von Hippel-Lindau tumor suppressor gene [Ivanov et al., Proc Natl Acad Sci (USA), 95: 12596-12601 (1998)]. Low levels of oxygen lead to stabilization of HIF-1α, which in turn leads to the increased expression of MN [Wykoff et al., Cancer Res, 60: 7075-7083 (2000)]. Areas of high expression of MN in cancers are linked to tumor hypoxia as reported in many cancers, and incubation of tumor cells under hypoxic conditions leads to the induction of MN expression [Wykoff et al., Cancer Res, 60: 7075-7083 (2000); Koukourakis et al., Clin Cancer Res, 7: 3399-3403 (2001); Giatromanolaki et al., Cancer Res, 61: 7992-7998 (2001); Swinson et al., J Clin Oncol, 21: 473-482 (2003); Chia et al., J Clin Oncol, 19: 3660-3668 (2001); Loncaster et al., Cancer Res, 61: 6394-6399 (2001)].
Key elements of the MN/CA9 promoter are the HIF-1 and SP1 binding regions [Kaluz et al., Cancer Res. 63: 917-922 (2003)] [PR1-HRE element]. The MN/CA9 promoter sequence (−3/−10) between the transcription start and PR1 contains a HRE element recognized by a hypoxia inducible factor 1 (HIF-1), which governs transcriptional responses to hypoxia [Wykoff et al., Cancer Res. 60: 7075-7083 (2000)]. The promoter of the CA9 gene contains five regions protected in DNase I footprinting (PR1-PR5, numbered from the transcription start) [Kaluz et al., J. Biol. Chem., 274: 32588-32595 (1999)]. PR1 and PR2 bind SP1/3 and AP1 transcription factors and are critical for the basic activation of CA9 transcription [Kaluz et al., J. Biol. Chem., 274: 32588-32595 (1999); Kaluzova et al., Biochem. J., 359: 669-677 (2001)]. HIF-1 strongly induces transcription of the CA9 gene in hypoxia, but for full induction requires a contribution of the SP1/3 transcription factor binding to PR1 [Wykoff et al., Cancer Res., 60: 7075-7083 (2000); Kaluz, et al., Cancer Res., 63: 917-922 (2003)].
Regulation under normoxia also requires SP1 [Kaluz et al., Cancer Res. 62: 4469-4477 (2002)]. Upregulation of CA9 transcription in increased cell density involves a mild pericellular hypoxia, depends upon cooperation of SP1 with HIF-1 at subhypoxic level and operates via the PI3K pathway [Kaluz et al., Cancer Res. 62: 4469-4477 (2002)]. Hypoxia and cell density act in an additive fashion so that the highest expression of CA9 is achieved under conditions of low oxygen at high density [Kaluz et al., Cancer Res., 62: 4469-4477 (2002)].
MN's Intracellular Region and the EGFR Pathway
As indicated above, CA9 expression is upregulated by both HIF-1α- and PI3K-dependent pathways, and both the PG and CA extracellular domains of CA IX have predicted roles in tumorigenesis based on cell adhesion and carbonic anhydrase activities. The invention disclosed herein is based on the discovery of a potential tumorigenic role of a third CA IX domain, the intracellular domain (IC). The inventor discloses finding CA IX to be associated with EGFR in lipid rafts in RCC cell lines, and that the sole tyrosine moiety of CA IX present in its IC domain can be phosphorylated in an EGFR-dependent manner. The inventor found evidence that tyrosine-phosphorylated CA IX interacts with the regulatory subunit of PI3K (p85), resulting in activation of Akt. That finding indicates that there is a positive feedback loop for CA9 expression in RCC, mediated by the PI3K pathway, which may contribute to the aggressiveness of RCC. Based on those novel findings, the instant invention discloses therapeutic methods targeted to the EGFR pathway which can be used alone, or in combination with other MN-targeted methods, to treat preneoplastic/neoplastic diseases characterized by abnormal MN expression.